Confirmation and Functional Characterization of Candidate Genes for Iron Toxicity Tolerance in Rice (Oryza sativa L.)
Plant Breeding and Plant Pathology
Final Report Abstract
Iron (Fe) toxicity is one of the major mineral disorders affecting rice production in flooded soils. Using genome mapping approaches we had previously identified five candidate genes for tolerance to Fe toxicity in rice based on physical positions and gene annotations related to Fe metabolism or stress response. This project aimed at advancing the confirmation and functional characterization of these genes. Knockout and knockdown mutants from public gene banks in Nipponbare (NB, japonica subspecies) or Dongjin (DJ, japonica subspecies) genetic background were characterized if available. In addition we employed CRISPR/cas9 technology to specifically mutate target genes, or over-expression genes in NB or IR64 (indica subspecies) genetic background. To this end, we newly established a rice genome editing and transformation pipeline. The involvement of a potassium ion channel gene (OsAKT1) in Fe toxicity tolerance was confirmed using two independent mutant lines (knockout KO, and knockdown KD) in NB background, which showed consistent sensitive phenotypes in terms of symptom formation. Mutants contained less potassium and more Fe in aboveground plant organs, which resulted in more significant damage of photosynthetic pigments compared to wildtypes (WT). These data conclusively demonstrated that potassium is involved in Fe translocation from roots to shoots in Fe toxic condition, and consequently affects Fe tolerance. For a glutathione-S-transferase gene (OsGST1) a single mutant line (KO) did not respond differently to Fe toxicity compared to WT. However, sequence analyses revealed substantial mutations of the IR64 allele of OsGST1, which could affect gene function and consequently Fe tolerance. Therefore, IR64 lines with mutated OsGST1 variants were generated by genome editing in addition to NB lines overexpressing OsGST1 from IR64. T0 plants with confirmed mutations or insertions were successfully recovered. A single knockout line in DJ background for a 4,5-DOPA dioxygenase extradiol gene (OsDODA1) was more sensitive to Fe toxicity than WTs in terms of symptom development and lipid peroxidation, and it showed enhanced chlorophyll degradation under stress. It also showed altered distribution of Fe within the shoot. Due to the promising results, additional mutant lines and over-expression lines were generated and positive T0 plants were confirmed. A KO line for a putative vacuolar ATPase (OsVATP1) showed stunted root growth and reduced tiller number. Also, more pronounced leaf bronzing symptoms and lipid peroxidation in shoots compared to WT indicated lower tolerance to Fe toxicity. In leaf blades, mutants showed significantly higher Fe concentration in Fe stress conditions. Due to the promising results, additional mutant lines and over-expression lines were generated and positive T0 plants were confirmed. For the last candidate gene, a putative casein kinase I (OsCK1), no viable KD or KO lines were identified from public gene banks. Therefore, new mutant lines were generated using genome editing and positive T0 plants were confirmed. In addition to these laboratory experiments, a field experiment was conducted in Madagascar. Twenty-three selected rice varieties representing local and exotic germplasm were grown in Fe toxic soil with and without the application of fertilizer in order to explore tolerance ranking and Fe responses. The experiments demonstrated, among others, that fertilizer application significantly affected Fe uptake, and that leaf bronzing symptoms, which are often employed as a phenotypic trait in the selection for Fe tolerance, were correlated with oxidative stress formation and biomass development. In summary, this project confirmed the involvement of several candidate genes in Fe tolerance in rice. In order to consolidate these findings, further plant materials for in-depth characterization were generated by genome editing and gene overexpression. Moreover, novel insights of Fe toxicity responses with different nutrient management strategies were obtained in field conditions. The project thus advanced our fundamental understanding of Fe metabolism in rice, but also has practical implications for crop production and breeding.
Publications
- (2017) Management and genotype effects on resistance to iron toxicity in lowland rice in Madagascar. Tropentag, 20th to 22nd September 2017. Bonn, Germany
Ergezinger L, Rajonandraina T, Wu LB, Rakotoson T, Frei M
- (2018) Adapting cereal crops to abiotic stresses. Invited lecture. XXII International Congress of Genetics, 10th to 14th September 2018, Foz do Iguaçu, Brazil
Frei M
- (2018) Confirmation and functional characterization of potassium ion channel gene for iron toxicity tolerance in rice. Tropentag, 17th to 19th September 2018, Ghent, Belgium
Holtkamp F, Wu LB, Frei M
- (2018) Iron toxicity tolerance in rice (Oryza sativa L.) – loci, mechanisms, and genes. XXII International Congress of Genetics, 10th to 14th September 2018, Foz do Iguaçu, Brazil
Wu LB, Holtkamp F, Frei M
